The long-term objective of this study is to determine the crystal structures of the remaining orphan nuclear receptor ligand-binding domains (LBDs) and to reveal the functional implications of these structures in their respective signaling pathways. Nuclear receptors are DNA-binding and ligand-dependent transcriptional factors that modulate gene expression involved in a broad spectrum of physiology. The LBD is the key structural domain that mediates the ligand signaling of nuclear receptors. In addition to ligand binding, the LBD contains dimerization motifs and a conserved surface that mediates ligand-dependent or independent recruitment of coactivators and corepressors for transcriptional regulation. The LBD has thus been the focus of intense structural studies and pharmaceutical discovery. Crystal structures of more than half of the 48 human nuclear hormone receptor LBDs have been determined and there are only a few orphan nuclear receptors for which LBD structure remains to be solved. The hypotheses of these applications are: 1) the specificity of diverse signaling pathways mediated by nuclear hormone receptors is in large part contained with n the structural components of their LBDs, and 2) the structure of each LBD will provide key information for understanding the molecular basis of ligand recognition, receptor dimerization, and protein-interacting surfaces that mediate specific signaling pathways by each receptor. Our specific aims are focused on crystallization and structural determination of the remaining orphan nuclear receptor LBDs, particularly, 1) the CAR LBD, 2) the COUP-TFI or -TFII LBD, 3) the TR2 or TR4 LBD, and 4) the SHP LBD. Following the structural determination, we will identify key structural elements by scrutinizing and analyzing the structures, and we will collaborate with Ming-Jer Tsai (Baylor College of Medicine), Steve Kliewer (University of Texas Southwestern Medical Center), and Doug Engel (University of Michigan), on site-directed mutagenesis and cell-based assays to validate the functional significance of these key structural elements. Significance: The structural information generated in this application will significantly enhance our understanding of the molecular mechanisms of how these orphan nuclear receptors have evolved for their respective ligand-dependent or -independent signaling pathways, and can serve as rational templates for drug discovery that targets these receptors.